1. Field of the invention
The present invention relates to the general field of microscopy and particularly the field of Fourier-transform infrared (FT-IR) microspectrophotometry.
2. Description of related art
The ready existence of commercial FT-IR spectrophotometers greatly facilitates infrared analysis of specimens. The utility of FT-IR microspectroscopy, however, is limited by the size of the microscopic sample that can be observed. If a sample is too small, radiant energy from surrounding areas reaches the detector and produces a spectrum containing the combined features of all materials in the field of view of the microscope. This phenomenon is termed spectroscopic mixing and is a problem which is particularly acute for an infrared microscope because the longer wavelength of the infrared radiation results in lower inherent resolution as compared to, for example, a visible light microscope. Spectroscopic mixing may be eliminated by electronically subtracting the spectrum of a known material from a mixed spectrum. Electronic subtraction does not work, however, without first knowing both the spectral features and the relative intensity of the features for the area immediately surrounding the object of interest. As a practical matter, electronic subtraction often does not work because the spectrum of the surrounding material cannot be isolated.
The resolution of any microscope is limited by the effects of diffraction. Diffraction depends on the wavelength of the radiant energy, the numeric aperture of the optical system and the spatial coherence of the radiation. The smallest separation of objects that may be resolved is typically expressed in terms of Rayleigh's criterion which is mathematically defined as 0.61 times the wavelength of the radiant energy divided by the numerical aperture of the microscope. Diffraction, however, has long been recognized as a practical limit to resolution and not as a theoretical limit. Only the wavelength of the radiant energy ultimately need limit the resolution of a microscope.
Minsky U.S. Pat. No. 3,013,476, discloses what has come to be known in the art as a confocal scanning microscope. The confocal microscope uses two pinhole apertures positioned at focal planes. One pinhole aperture is placed between a light source and an objective lens at a real image plane so that the lens focuses the light emerging from the pinhole aperture onto a sample. A second pinhole aperture is positioned between an objective lens and a detector at a real image plane so that light from the sample is focused onto the second pinhole aperture. A sample is placed at the sample image plane and moved in a scanning pattern so that the detector supplies an input signal corresponding to the raster scan of a television. Minsky discloses that the confocal scanning microscope reduces the depth of field for an optically thick transmissive sample because the detector receives very little light from outside the plane of the sample image plane.
Others have noted that a confocal microscope increases the image contrast by reducing the amount of stray light that reaches the detector from outside the image plane. This superior depth of field resolution in a confocal microscope produces an apparent increase in resolution. Sheppard and Wilson of the University of Oxford have noted that a scanning confocal microscope may obtain an actual increase in the resolution above that anticipated for using the same optics without a confocal arrangement. This actual improvement in resolution, however, is only as great as a factor of 2.4 and is accompanied by a reduction in image contrast and, hence, a reduction in apparent resolution.
Experimental research in the field of confocal microscopy apparently has been directed to producing a scanning microscope for forming an image of a sample. The resolution of a scanning microscope is limited by the motion of the sample relative to the radiant energy. No known experiment has attempted to determine the actual point to point resolution of a confocal microscope.
Present mathematical models of image formation in a confocal microscope provide an inadequate explanation of image formation in the field of microspectrophotometry. The transform function for a confocal microscope involves a complicated convolution of the transform functions of the condensing and collecting optics as well as the focal plane apertures. The only exact mathematical solutions that have been published involve the special cases of point to point imaging of a point source that emits radiant energy that is either perfectly coherent or perfectly incoherent. Microspectrophotometry, however, necessarily involves the use of partially coherent radiant energy. The source of the radiant energy has effective spatial dimensions that are determined by the size and shape of the sample. The wavelength range of the radiant energy is determined by the wavelength range of the spectroscopic characteristics of the sample which are to be observed. The optical properties of a confocal microscope that uses partially coherent radiant energy is therefore important for microspectrophotometry and must be determined experimentally.
Practical concerns relating to throughput efficiency mitigate against using a confocal microscope in microspectroscopy. Conventional confocal microscopes have used a laser as an intense light source to supply monochromatic light to the source side pinhole aperture. However, conventional spectrometers, especially FT-IR spectrophotometers, do not have an intense source of constant amplitude radiant energy over a broad frequency range. Further, confocal microscopes have only attempted to image samples and not to distinguish between regions on a sample having different compositions. Thus, the theory of confocal microscopy and conventional confocal scanning microscopes have held no practical utility for microspectroscopy in general or for FT-IR microspectrophotometry in particular.